scholarly journals Development and Evaluation of a Loop-Mediated Isothermal Amplification Method for Rapid Detection of Aspergillus fumigatus

2016 ◽  
Vol 54 (4) ◽  
pp. 950-955 ◽  
Author(s):  
Qing Tang ◽  
Shuguang Tian ◽  
Nong Yu ◽  
Xi Zhang ◽  
Xiaodong Jia ◽  
...  
Keyword(s):  
Aspergillus Fumigatus ◽  
Rapid Detection ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Clinical Samples ◽  
Content Type ◽  
Loop Mediated Isothermal Amplification ◽  
Real Time Quantitative Pcr ◽  
Amplification Method ◽  
Positive Results

Aspergillus fumigatusis a conditional pathogen and the major cause of life-threatening invasive aspergillosis (IA) in immunocompromised patients. The early and rapid detection ofA. fumigatusinfection is still a major challenge. In this study, the new member of the fungal annexin family, annexin C4, was chosen as the target to design a loop-mediated isothermal amplification (LAMP) assay for the rapid, specific, and sensitive detection ofA. fumigatus. The evaluation of the specificity of the LAMP assay that was developed showed that no false-positive results were observed for the 22 non-A. fumigatusstrains, including 5 species of theAspergillusgenus. Its detection limit was approximately 10 copies per reaction in reference plasmids, with higher sensitivity than that of real-time quantitative PCR (qPCR) at 102copies for the same target. Clinical samples from a total of 69 patients with probable IA (n =14) and possible IA (n= 55) were subjected to the LAMP assay, and positive results were found for the 14 patients with probable IA (100%) and 34 patients with possible IA (61.82%). When detection using the LAMP assay was compared with that using qPCR in the 69 clinical samples, the LAMP assay demonstrated a sensitivity of 89.19% and the concordance rate for the two methods was 72.46%. Accordingly, we report that a valuable LAMP assay for the rapid, specific, and simple detection ofA. fumigatusin clinical testing has been developed.

scholarly journals Rapid Detection of Clostridium botulinum in Food Using Loop-Mediated Isothermal Amplification (LAMP)

2021 ◽  
Vol 18 (9) ◽  
pp. 4401
Author(s):  
Yufei Chen ◽  
Hao Li ◽  
Liu Yang ◽  
Lei Wang ◽  
Ruyi Sun ◽  
...  
Keyword(s):  
Rapid Detection ◽  
High Throughput Screening ◽  
Clostridium Botulinum ◽  
Lamp Assay ◽  
High Specificity ◽  
Isothermal Amplification ◽  
Clinical Samples ◽  
Loop Mediated Isothermal Amplification ◽  
Specificity And Sensitivity ◽  
Target Dna

Botulinum neurotoxins are considered as one of the most potent toxins and are produced by Clostridium botulinum. It is crucial to have a rapid and sensitive method to detect the bacterium Clostridium botulinum in food. In this study, a rapid detection assay of C. botulinum in food using loop-mediated isothermal amplification (LAMP) technology was developed. The optimal primers were identified among three sets of primers designed specifically based on the partial ntnh gene encoding nontoxic-nonhaemagglutinin (NTNH) for rapid detection of the target DNA in plasmids. The optimal temperature and reaction time of the LAMP assay were determined to be 64 °C and 60 min, respectively. The chemical kit could be assembled based on these optimized reaction conditions for quick, initial high-throughput screening of C. botulinum in food samples. The established LAMP assay showed high specificity and sensitivity in detecting the target DNA with a limit of 0.0001 pg/ul (i.e., ten times more sensitive than that of the PCR method) and an accuracy rate of 100%. This study demonstrated a potentially rapid, cost-effective, and easy-operating method to detect C. botulinum in food and clinical samples based on LAMP technology.

scholarly journals Rapid Detection of Viable Salmonellae in Produce by Coupling Propidium Monoazide with Loop-Mediated Isothermal Amplification

2011 ◽  
Vol 77 (12) ◽  
pp. 4008-4016 ◽  
Author(s):  
Siyi Chen ◽  
Fei Wang ◽  
John C. Beaulieu ◽  
Rebecca E. Stein ◽  
Beilei Ge
Keyword(s):  
Pure Culture ◽  
Rapid Detection ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Detection Methods ◽  
Sample Treatment ◽  
Propidium Monoazide ◽  
Simple Method ◽  
Content Type ◽  
Loop Mediated Isothermal Amplification

ABSTRACTRecent outbreaks linked toSalmonella-contaminated produce heightened the need to develop simple, rapid, and accurate detection methods, particularly those capable of determining cell viability. In this study, we examined a novel strategy for the rapid detection and quantification of viable salmonellae in produce by coupling a simple propidium monoazide sample treatment with loop-mediated isothermal amplification (PMA-LAMP). We first designed and optimized a LAMP assay targetingSalmonella. Second, the performance of PMA-LAMP for detecting and quantifying viable salmonellae was determined. Finally, the assay was evaluated in experimentally contaminated produce items (cantaloupe, spinach, and tomato). Under the optimized condition, PMA-LAMP consistently gave negative results for heat-killedSalmonellacells with concentrations up to 108CFU/ml (or CFU/g in produce). The detection limits of PMA-LAMP were 3.4 to 34 viableSalmonellacells in pure culture and 6.1 × 103to 6.1 × 104CFU/g in spiked produce samples. In comparison, PMA-PCR was up to 100-fold less sensitive. The correlation between LAMP time threshold (TT) values and viableSalmonellacell numbers was high (R2= 0.949 to 0.993), with a quantification range (102to 105CFU/reaction in pure culture and 104to 107CFU/g in produce) comparable to that of PMA in combination with quantitative real-time PCR (PMA-qPCR). The complete PMA-LAMP assay took about 3 h to complete when testing produce samples. In conclusion, this rapid, accurate, and simple method to detect and quantify viableSalmonellacells in produce may present a useful tool for the produce industry to better control potential microbial hazards in produce.

scholarly journals Development and Validation of a Loop-Mediated Isothermal Amplification (LAMP) Assay for Rapid Detection of Glaesserella (Haemophilus) parasuis

2020 ◽  
Vol 9 (1) ◽  
pp. 41
Author(s):  
Veronika Pilchová ◽  
Diana Seinige ◽  
Isabel Hennig-Pauka ◽  
Kathrin Büttner ◽  
Amir Abdulmawjood ◽  
...  
Keyword(s):  
Respiratory Tract ◽  
Rapid Detection ◽  
Limit Of Detection ◽  
Colorimetric Assay ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Economic Losses ◽  
Clinical Samples ◽  
Analytical Sensitivity ◽  
Loop Mediated Isothermal Amplification

Glaesserella parasuis is a fastidious pathogen that colonizes the respiratory tract of pigs and can lead to considerable economic losses in pig production. Therefore, a rapid detection assay for the pathogen, preferably applicable in the field, is important. In the current study, we developed a new and improved detection method using loop-mediated isothermal amplification (LAMP). This assay, which targets the infB gene, was tested on a collection of 60 field isolates of G. parasuis comprising 14 different serovars. In addition, 63 isolates from seven different closely related species of the family Pasteurellaceae, including A. indolicus, A. porcinus, and A. minor, and a species frequently found in the respiratory tract of pigs were used for exclusivity experiments. This assay showed an analytical specificity of 100% (both inclusivity and exclusivity) and an analytical sensitivity of 10 fg/µL. In further steps, 36 clinical samples were tested with the LAMP assay. An agreement of 77.1 (95% CI: 59.9, 89.6) and 91.4% (95% CI: 75.9, 98.2) to the culture-based and PCR results was achieved. The mean limit of detection for the spiked bronchoalveolar lavage fluid was 2.58 × 102 CFU/mL. A colorimetric assay with visual detection by the naked eye was tested to provide an alternative method in the field and showed the same sensitivity as the fluorescence-based LAMP assay. Overall, the optimized LAMP assay represents a fast and reliable method and is suitable for detecting G. parasuis in the laboratory environment or in the field.

Rapid detection of Betacoronavirus 1 from clinical fecal specimens by a novel reverse transcription loop-mediated isothermal amplification assay

2011 ◽  
Vol 24 (1) ◽  
pp. 174-177 ◽  
Author(s):  
Jun Qiao ◽  
Qingling Meng ◽  
Xuepeng Cai ◽  
Chuangfu Chen ◽  
Zaichao Zhang ◽  
...  
Keyword(s):  
Reverse Transcription ◽  
Rapid Detection ◽  
Lamp Assay ◽  
High Specificity ◽  
Isothermal Amplification ◽  
Clinical Samples ◽  
Lamp Method ◽  
Nucleocapsid Gene ◽  
Loop Mediated Isothermal Amplification ◽  
Amplification Assay

Betacoronavirus 1 (BCoV-1) is an important pathogen causing diarrhea in calves. In the current study, a novel reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for rapid detection of BCoV-1 was successfully developed. The primers were designed to target the highly conserved fragment of BCoV-1 nucleocapsid gene. The assay displayed high specificity detecting only BCoV-1 with no cross reaction with other viruses. When 418 clinical samples from 6 different geographical areas of Xinjiang province were tested by the RT-LAMP method, the results indicated that this test is a simple, rapid, accurate, and sensitive method for the detection of BCoV-1.

scholarly journals A Multiplex and Colorimetric Reverse Transcription Loop-Mediated Isothermal Amplification Assay for Sensitive and Rapid Detection of Novel SARS-CoV-2

2021 ◽  
Vol 11 ◽  
Author(s):  
Eduardo Juscamayta-López ◽  
Faviola Valdivia ◽  
Helen Horna ◽  
David Tarazona ◽  
Liza Linares ◽  
...  
Keyword(s):  
Reverse Transcription ◽  
Rapid Detection ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Clinical Samples ◽  
Molecular Testing ◽  
Respiratory Pathogens ◽  
Loop Mediated Isothermal Amplification ◽  
A Genome ◽  
Assay Precision

Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has become a major threat to public health. Rapid molecular testing for convenient and timely diagnosis of SARS-CoV-2 infections represents a challenge that could help to control the current pandemic and prevent future outbreaks. We aimed to develop and validate a multiplex and colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay using lyophilized LAMP reagents for sensitive and rapid detection of SARS-CoV-2. LAMP primers were designed for a set of gene targets identified by a genome-wide comparison of viruses. Primer sets that showed optimal features were combined into a multiplex RT-LAMP assay. Analytical validation included assessment of the limit of detection (LoD), intra- and inter-assay precision, and cross-reaction with other respiratory pathogens. Clinical performance compared to that of real-time reverse transcriptase-polymerase chain reaction (RT-qPCR) was assessed using 278 clinical RNA samples isolated from swabs collected from individuals tested for COVID-19. The RT-LAMP assay targeting the RNA-dependent RNA polymerase (RdRp), membrane (M), and ORF1ab genes achieved a comparable LoD (0.65 PFU/mL, CT=34.12) to RT-qPCR and was 10-fold more sensitive than RT-qPCR at detecting viral RNA in clinical samples. Cross-reactivity to other respiratory pathogens was not observed. The multiplex RT-LAMP assay demonstrated a strong robustness and acceptable intra- and inter-assay precision (mean coefficient of variation, 4.75% and 8.30%). Diagnostic sensitivity and specificity values were 100.0% (95% CI: 97.4–100.0%) and 98.6% (95% CI: 94.9–99.8%), respectively, showing high consistency (Cohen’s kappa, 0.986; 95% CI: 0.966–1.000; p<0.0001) compared to RT-qPCR. The novel one-step multiplex RT-LAMP assay is storable at room temperature and showed similar diagnostic accuracy to conventional RT-qPCR, while being faster (<45 min), simpler, and cheaper. The new assay could allow reliable and early diagnosis of SARS-CoV-2 infections in primary health care. It may aid large-scale testing in resource-limited settings, especially if it is integrated into a point-of-care diagnostic device.

scholarly journals Development and validation of a one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP) for rapid detection of ZIKV in patient samples from Brazil

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Severino Jefferson Ribeiro da Silva ◽  
Keith Pardee ◽  
Udeni B. R. Balasuriya ◽  
Lindomar Pena
Keyword(s):  
Reverse Transcription ◽  
Rapid Detection ◽  
Quantitative Polymerase Chain Reaction ◽  
Rna Extraction ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Serum Samples ◽  
Low Resource ◽  
Loop Mediated Isothermal Amplification ◽  
One Step

AbstractWe have previously developed and validated a one-step assay based on reverse transcription loop-mediated isothermal amplification (RT-LAMP) for rapid detection of the Zika virus (ZIKV) from mosquito samples. Patient diagnosis of ZIKV is currently carried out in centralized laboratories using the reverse transcription-quantitative polymerase chain reaction (RT-qPCR), which, while the gold standard molecular method, has several drawbacks for use in remote and low-resource settings, such as high cost and the need of specialized equipment. Point-of-care (POC) diagnostic platforms have the potential to overcome these limitations, especially in low-resource countries where ZIKV is endemic. With this in mind, here we optimized and validated our RT-LAMP assay for rapid detection of ZIKV from patient samples. We found that the assay detected ZIKV from diverse sample types (serum, urine, saliva, and semen) in as little as 20 min, without RNA extraction. The RT-LAMP assay was highly specific and up to 100 times more sensitive than RT-qPCR. We then validated the assay using 100 patient serum samples collected from suspected cases of arbovirus infection in the state of Pernambuco, which was at the epicenter of the last Zika epidemic. Analysis of the results, in comparison to RT-qPCR, found that the ZIKV RT-LAMP assay provided sensitivity of 100%, specificity of 93.75%, and an overall accuracy of 95.00%. Taken together, the RT-LAMP assay provides a straightforward and inexpensive alternative for the diagnosis of ZIKV from patients and has the potential to increase diagnostic capacity in ZIKV-affected areas, particularly in low and middle-income countries.

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